Self-assembling complex for targeting chemical agents to cells

ABSTRACT

The present invention relates to a complex that can be injected into the body to hone in on target cells to deliver molecules. In one embodiment, the invention provides a drug delivery system that includes components that self-assemble into one targeted conjugate. In another embodiment, the invention includes a targeted carrier protein and a nucleic acid sequence non-covalently linked to one or more drugs.

RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 12/667,436,filed on Dec. 31, 2009, which in turn is an application filed under 35U.S.C. §371 based on the International Application PCT/US2008/069239filed on Jul. 3, 2008, which in turn claims priority to the U.S.Provisional Application Ser. No. 60/948,242, filed on Jul. 6, 2007, allof which applications are incorporated by reference herein in theirentirety, including the drawings.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.CA129822, Grant No. CA102126, and Grant No. CA116014 awarded by NationalInstitutes of Health. The government has certain rights in theinvention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of biotechnology; specifically, todelivery systems for chemical agents.

BACKGROUND OF THE INVENTION

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Current strategies for targeting therapy to tumors includeantibody-targeted chemotherapy agents (i.e., immunoconjugates), targetedtoxins, signal-blocking antibodies, and antibody-targeted liposomes(i.e., immunoliposomes). In fact, HER2+ breast tumors, whichover-express subunit 2 of the human epidermal growth factor receptor(HER), comprise a significant subset of breast cancers that arerecalcitrant to standard methods of treatment, and predict a highmortality. The abnormally high level of HER on the surface of thesetumor cells may enable targeted therapeutics to home in on these cells,thus making HER2+ tumors ideal candidates for targeted therapy. However,the aforementioned therapies are problematic—with respect to HER2+breast tumors and other forms of cancer—because they require chemicalmodification which may be costly and can impair activity of the drugand/or the carrier; may use recombinant antibodies that can losestructure in physiological conditions and thus result in impairedtargeting activity; are not able to penetrate into the cell; focus onthe need to modulate receptor signaling which can be impaired in tumorcells; and have off-target effects such as heart toxicity.

Immunoconjugate therapies rely on the chemical coupling of single-chainantibodies to drugs, whereby the antibody directs the drug to specificcells by recognizing certain cell surface proteins or receptors [K. A.Chester et al., Disease Markers, 16:53-62 (2000); A. E. Frankel et al.,Clin. Cancer Res., 6:326-334 (2000)]. Studies have shown, however, thatsuch antibodies can unfold and aggregate at physiological temperature,which would impede binding to target cells, and result in lowtherapeutic efficacy [R. Glockshuber et al., Biochem, 29:1362-1367(1990); M. Schmidt et al., Oncogene, 18:1711-1721 (1999)]. Moreover,covalent linkage of drug to carrier can impair the activity of bothmolecules, as well as entail high production cost.

In contrast, studies in connection with one embodiment of the presentinvention show that an exemplary targeted carrier protein, HerPBK10,retains receptor targeting under physiological conditions in thepresence of serum, indicating that the targeted carrier does not unfoldor lose receptor binding [H. Agadjanian et al., Pharm Res., 23:367-377(2006)]. Furthermore, the present invention is engineered so that thedrug self-assembles with the carrier molecule and thus does not requirechemical modifications to covalently link the molecules together. Thisnon-covalent assembly thus allows both the targeted carrier and drug toremain unmodified and thus preserve structure and activity. Finally,recombinant proteins (such as HerPBK10) can be produced in bulkquantities by large-scale fermentation for a lower cost compared tomonoclonal antibody generation and production.

An alternative approach to tumor targeting has been the development oftoxic proteins, such as plant or bacterial toxins, that are modified byappendage to a targeting peptide (or ligand) [A. E. Frankel et al.,Clin. Cancer Res., 6:326-334 (2000)]. Such proteins are produced byrecombinant methods (i.e., genes are engineered to produce the proteinsin a cell system, from which the proteins can then be isolated), and theresulting protein is a fusion of the toxin to the ligand. While fusiontoxin proteins can be produced by large scale fermentation as a moreefficient and lower cost alternative to antibody generation, the toxinrequires special processing to be active, thus limiting potency [M.Schmidt et al., Oncogene, 18:1711-1721 (1999); M. Jeschke et al., Intl.J. Cancer, 60:730-739 (1995)]. For example, the activity of recombinantdiptheria toxin transmembrane domain, used to enhance non-viral genetransfer, is reduced by as much as 75% when fused to a foreign peptide,indicating that appending a peptide to a toxin disables toxin activity[K. J. Fisher and J. M. Wilson, Biochem. J., 321:49-58 (1997)]. Thus,delivery by non-covalent means (i.e., self-assembly), which is a featureof the present invention, is believed to be advantageous in terms ofretaining drug potency.

Antibodies directed at the extracellular domain of HER2 have been usedto target drug complexes to the HER2 subunit, but have not necessarilyinduced internalization of the drug complex; thus limiting potency [D.Goren et al., Br. J. Cancer, 74:1749-1756 (1996)]. Such findingsillustrate that targeting is not enough, as a lack of targeted uptakecan limit efficacy. Alternatively, signal blocking antibodies have beendeveloped to inhibit the proliferative signal transduced throughoverexpression and high cell surface display of HER2 [J. Baselga et al.,J. Clin. Oncol., 14:737-744 (1996); M. A. Cobleigh et al., Proc. Am.Soc. Clin. Oncol., 17:97a (1998)]. One currently used targeted therapy,trastuzumab (Herceptin), an antibody directed against the HER2 subunit,blocks normal signaling but has been ineffective in about 70% of treatedpatients, possibly due to aberrant intracellular pathways in tumor cellsthat may not respond to signal inhibition [C. L. Vogel et al., “J. Clin.Oncol., 20:719-726 (2002); T. Kute et al., Cytometry, A57:86-93 (2004)].Furthermore, an ongoing concern with trastuzumab is the exquisitesensitivity of heart tissue to HER2 signal inhibition, which is furtherexacerbated by anthracycline chemotherapy agents [D. J. Slamon et al.,N. Engl. J. Med., 344:783-792 (2001)]. In one embodiment, the approachof the present invention instead takes advantage of the bindinginteraction of the natural ligand for HER, which has a greatly increasedligand affinity when HER2 is overexpressed. It is believed that this islikely to translate to lower, and thus safer, doses of drug whentargeted. Accordingly, the targeting approach of the present inventionshould avoid binding to tissues displaying low to normal receptorsubunit levels but exhibit preferential binding to HER2+ tumor cells.Moreover, the inventor has shown that the receptor binding domain ofheregulin that is incorporated into HerPBK10 induces rapidinternalization after binding to the heregulin receptor [L. K.Medina-Kauwe et al., BioTechniques, 29:602-609 (2000); L. K.Medina-Kauwe and X. Chen, Vitamins and Hormones, Elsevier Science, G.Litwack (Ed.), San Diego, 81-95 (2002)]; enabling uptake of DNA [L. K.Medina-Kauwe et al., Gene Ther., 8:1753-1761 (2001)] and fluorescentcompounds [H. Agadjanian et al., Pharm Res., 23:367-377 (2006)]. Theinventive approach, therefore, circumvents the need to modulate receptorsignaling, by exploiting the rapid receptor endocytosis induced byligand binding and the cytosolic penetration features of viral capsidprotein to directly transport drugs into the cell and inducecytotoxicity from within.

While targeted uptake facilitates drug entry into target cells, theintracellular disposition of the drug can still affect potency.Targeting antibodies delivering covalently linked drugs can betrafficked to lysosomes, thus sequestering the drug from subcellulartargets and limiting potency. Approaches to circumventing this includelinking the drug to a targeting antibody via an acid labile bond tofacilitate release into the endocytic compartment [P. A. Trail et al.,Cancer Immunol Immunother., 52:328-337 (2003); P. A. Trail et al.,Cancer Res., 52:5693-5700 (1992); P. A. Trail et al., Science,261:212-215 (1993); G. R. Braslawsky et al., Cancer Res., 50:6608-6614(1990)]. However, bond instability can reduce in vivo potency, likely bycausing premature drug release and thus delivery to non-target tissue.In contrast, the endosomal disruption feature of one embodiment of thepresent invention has the advantage of endosomal escape, thusfacilitating release of the drug into the cell cytoplasm and access tointracellular targets, which can include passengering of nucleartargeted molecules and passage through nuclear pores.

Immunoliposomes, carrying doxorubicin (“Dox”) and targeted to HER2, havebeen developed and can accumulate in tumor tissue in animal models [J.W. Park et al., Clin. Cancer Res., 8:1172-1181 (2002)], likely due tothe leaky tumor vasculature D. C. Drummond et al., Pharmacol. Rev.,51:691-743 (1999)]. Release of Dox from these liposomes is thought tooccur via the acidic tumor environment, lipase release from dying cells,and enzyme and oxidizing agent release from infiltrating inflammatorycells [G. Minotti et al., Pharmacol. Rev., 56:185-229 (2004)]. Theseconditions may induce premature drug release and nonspecific delivery,though the accumulation of immunoliposomes at tumor sites may tend tofavor drug uptake at the tumor. Studies using the trastuzumab Fab′fragment for liposome targeting of Dox have demonstrated antitumorefficacy [J. W. Park et al., Clin. Cancer Res., 8:1172-1181 (2002],though the effect on cardiac tissue was not reported in that study. Incontrast, for reasons described above, it is believed that in anembodiment, the inventive system yields effective targeted drug deliverydue to high affinity receptor-ligand binding and rapid endocytosiscoupled with the membrane-penetrating activity of the viral penton baseprotein to ensure delivery into target cells.

SUMMARY OF THE INVENTION

Various embodiments provide a drug delivery molecule, comprising apolypeptide sequence adapted to target and/or penetrate a type of cell,a nucleic acid sequence bound to the polypeptide sequence viaelectrostatic interactions, and a chemical agent non-covalently linkedto the nucleic acid sequence. In other embodiments, the polypeptidesequence comprises a targeting ligand, an endosomolytic domain, apositively charged domain, and/or a polylysine motif. In otherembodiments, the chemical agent is doxorubicin, or a pharmaceuticallyequivalent thereof. In other embodiments, the type of cell is a HER2+breast cancer cell.

Other embodiments provide a self-assembling complex, comprising arecombinant fusion protein, and a double-stranded oligonucleotide, boundto the recombinant fusion protein by electrostatic interactions. Inanother embodiment, the recombinant fusion protein comprises a Hersegment. In another embodiment, the recombinant fusion protein comprisesa penton base segment. Various embodiments also include a decalysinesegment.

Other embodiments also include a method of preparing a drug deliverymolecule, comprising the following steps of incubating an intercalatingdrug with a polynucleotide sequence to create a complex, and incubatingsaid complex with a targeted carrier protein to form the drug deliverymolecule. In another embodiment, the intercalating drug is doxorubicin,or a pharmaceutically equivalent thereof. Various other embodimentsprovide for the polynucleotide sequence to be double-stranded. In otherembodiments, the targeted carrier protein comprises a targeting ligand.In another embodiment, the targeting ligand comprises the receptorbinding domain of heregulin-α. In another embodiment, the targetedcarrier protein contains an endosomolytic domain. In another embodiment,the endosomolytic domain comprises an Arg-Gly-Asp motif. In anotherembodiment, the endosomolytic domain comprises a Glu-Gly-Asp motif. Inother embodiments, the targeted carrier protein comprises a polylysinemotif. In other embodiments, the polylysine motif is a decalysine. Inother embodiments, the polynucleotide sequence is SEQ. ID. NO.: 6, SEQ.ID. NO.: 7, SEQ. ID. NO.: 8, SEQ. ID. NO.: 9, or a combination thereof.

Various embodiments provide methods of treating a disease in anindividual, comprising providing a drug delivery molecule comprising apolypeptide sequence adapted to target and/or penetrate a type of cell,a nucleic acid sequence bound to the polypeptide sequence viaelectrostatic interactions, a chemical agent non-covalently linked tothe nucleic acid sequence, and administering a therapeutically effectiveamount of the drug delivery molecule to the individual to treat thedisease. In another embodiment, the disease is breast cancer. In otherembodiments, the chemical agent is a chemotherapeutic agent. In otherembodiments, the chemical agent is doxorubicin, or a pharmaceuticallyequivalent thereof. In other embodiments, the targeted carrier proteincomprises a targeting ligand, an endosomolytic domain, and/or apolylysine motif. In other embodiments, the individual is a human. Inother embodiments, the individual is a mouse. In other embodiments, thetype of cell is a glioma cell. In other embodiments, the polypeptidesequence is PBK10. In other embodiments, the disease is metastaticcancer.

Other embodiments include a composition comprising a drug deliverymolecule comprising a polypeptide sequence adapted to target and/orpenetrate a type of cell, a nucleic acid sequence bound to thepolypeptide sequence via electrostatic interactions, and a chemicalagent non-covalently linked to the nucleic acid sequence, and a carrier.In another embodiment, the polypeptide sequence comprises anendosomolytic domain.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousembodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 illustrates a delivery system in accordance with an embodiment ofthe present invention.

FIG. 2 illustrates a delivery system configured for the delivery of Doxto HER2+ breast cancer cells in accordance with an embodiment of thepresent invention. Step (1) illustrates HERPBK10 produced and purifiedas a recombinant fusion protein in bacterial. Step (2) illustratesDNA-Dox formed by noncovalent intercalation interaction. Step (3)illustrates DNA-Dox can bind HerPBK10 by noncovalent charge interaction(anionic DNA phosphates electrophillicaly bind cationic polylysine).

FIG. 3 illustrates a schematic of the operation of a delivery systemconfigured for the delivery of Dox to HER2+ breast cancer cells inaccordance with an embodiment of the present invention, including (1)receptor binding at the cell membrane, (2) internalization of thecomplex, (3) cytosolic release of the chemotherapeutic (Dox)non-covalently bonded to dsDNA via intercalation interactions, and (4)nuclear entry of the chemotherapeutic and dsDNA.

FIG. 4 (A)-(B) illustrates DNA-Dox assembly. The ds-oligo (prepared byannealing complimentary 30 bp oligonucleotides) was incubated 1 h atroom temp with Dox at 1:16 molar ratio DNA:Dox. Free Dox was removed byfiltration through 10 MW cutoff spin columns. FIG. 4 (A) illustratesrelative Dox in filtrates. After the first Dox removal spin (Wash 1) thefilters were washed 4 more times with HEPES buffered saline (HBS)(Washes 2-5). FIG. 4 (B) illustrates UV/V absorbances of filtrates andretentates.

FIG. 5 illustrates HerDox assembly. DNA-Dox was incubated with HerPBK10on ice for 2 h at 9:1 molar ratio HerPBK10:DNA-Dox. The mixture wassubject to size exclusion HPLC and fractions collected at minutes 6-10for SDS-PAGE and immunoblotting. In future experiments, HerDox iscollected from the 6 min peak. The concentration of Dox in HerDox wasassessed by measuring absorbance at 480 nm (Dox absorbance wavelength).HPLC purification of HerDox fractions 1-5 correspond to samplescollected at minutes 6-10. Immunoblot of fractions 1-5 is also depictedwith a penton base antibody used to identify HerPBK10.

FIG. 6 (A)-(B) illustrates conjugate stability under different storageconditions, or in serum. As illustrated in FIG. 6 (A), HerDox wasincubated up to 12 days at 4° C., RT, or 37° C. Samples were filteredthrough ultrafiltration spin columns every other day, and retentate andfiltrate absorbances measured at 480 nm to determine relative Doxretention or release from conjugates, respectively. As illustrated inFIG. 6 (B), to mimic extended exposure to cells in culture medium,HerDox immobilized on Ni-NTA beads were incubated with bovine serum inDMEM for the indicated times at 37° C. before each sample was pelleted.Absorbance of supernatants (Filtrates) and bead eluates (Retentates)were measured at 480 nm to detect Dox. Relative Dox release or retentionin serum is expressed as normalized to control (corresponding samplelacking serum). N=3 per time point. T tests (P≦0.05) of samples comparedto controls showed no significant differences.

FIG. 7 illustrates targeted toxicity. Each cell line was exposed toHerDox (0.5 uM Dox conc), Dox alone (0.5 uM), or HerPBK10 alone for 4 hat 37° C. in complete (i.e. serum-containing) media, followed byaspiration to remove free conjugate, and addition of fresh medium whilecells are grown continuously. Cell titer was determined by metabolic(i.e. MTT) assay. FIG. 7 upper panel illustrates the effect of HerDox onMDA-MB-231 (HER2−) and MDA-MB-435 (HER2+) cell survival. Relativesurviving cell numbers are represented as a % of corresponding untreatedcells. FIG. 7 lower panel illustrates comparison of HerDox, Dox alone,or HerPBK10 alone on HER2− and HER2+ cell survival. Relative survival(as % of untreated cells) is shown for Day 3 of treatment.

FIG. 8 illustrates receptor specificity. MDA-MB-435 (HER2+) cells wereincubated with free ligand (eHRG) at 10× molar excess of HerDox for 1 hat 4° C. Media was aspirated to remove free eHRG and fresh mediacontaining HerDox (0.5 uM) was added to cells. Cell survival wasmeasured by MTT assay and represented as a % of relative untreated cellnumbers.

FIG. 9 (A)-(C) illustrates targeting in a mixed cell culture. Asillustrated in FIG. 10 (A), equal numbers of MDA-MB-435 and GFP(+)MDA-MB-231 cells were treated with Dox alone (0.5 uM), Her-Dox(containing 0.5 uM Dox), or HerPBK10 (1.2 ug/well, equivalent toHerPBK10 in HerDox). Wells were assayed for GFP fluorescence (todetermine relative MDA-MB-231 number) and crystal violet staining (todetermine total cell number). As illustrated in FIG. 9 (B), cellsurvival was determined by calculating the relative doubling time (DT)of experimental (exp) cells normalized by control (con) cells based onthe crystal violet stains (total cells) and GFP fluorescence (MDA-MB-231cells). The DT of MDA-MB-435 was determined by subtracting the DT ofMDA-MB-231 from the total cell DT. Relative survival is shown for Day 2of treatment. As illustrated in FIG. 9 (C), there is stability in cellculture. Aliquots of culture media containing HerDox (after incubationfor the indicated times at 37° C.) were electrophoresed on a 2% agarosegel. HerDox incubated at 37° C. in HEPES-buffered saline, lacking serum,was processed in parallel. Dox fluorescence was visualized by UVexcitation. Free Dox (Dox alone) is not retained in the gel whereas Doxincorporated in HerDox is. To align fluorescent bands with HerPBK10 andassess loading per lane, the gel was stained with Coomassie blue, whichalso identified serum protein from culture media samples.

FIG. 10 illustrates preferential targeting of GFP-Her to HER2+ tumors.Tumor-bearing mice were injected with 3 nmoles of GFP-Her via the tailvein. Tissues were harvested at 3.5 h after injection and visualizedusing a Xenogen small-animal imager. GFP fluorescence is pseudo-coloredred (blue pseudo-coloring indicates no fluorescence whereas GFPintensity is reflected by a color value shift toward red in the colorbar).

FIG. 11 (A)-(B) illustrates preferential targeting of HerDox to HER2+tumors. Tumor-bearing mice were injected with 0.75 nmoles of HerDox orDox via the tail vein and imaged with a custom small animal imager. FIG.11 (A) depicts imaging of live mice after IV delivery of HerDox. Tumorsare indicated by arrows. FIG. 11 (B) depicts imaging of tumors andtissues harvested at 3 h after injection of HerDox or Dox. Fluorescencesignal from Dox is pseudo-colored according to the color bar, with ashift toward 100 indicating high fluorescence intensity. FIG. 11 (B)illustrates a comparison of the targeted delivery of Dox to HER2+ breastcancer cells with minimal delivery to other organs and tissues using adelivery system in accordance with an embodiment of the presentinvention (left panel), as compared with Dox administered alone (rightpanel).

FIG. 12 (A)-(D) illustrates comparison of HerDox and Dox on (A) tumorgrowth, (B) animal weight, (C) cardiac tissue, and (D) cardiac function.

FIG. 13 illustrates stability in mouse whole blood. Freshly collectedwhole blood was processed by ultrafiltration through 10K MW cutoffmembranes after up to 1 h incubation with HerDox or Dox at 37° C. As 0.5mM EDTA was used as an anticoagulant, HerDox in EDTA alone (-blood) wasprocessed in parallel. Bars represent fluorescence of retained(retentates) or released (filtrates) Dox as a percentage of the totalfluorescence of each sample. Scale of Y-axis is adjusted to showpresence of filtrate samples. N=3 per treatment.

FIG. 14 (A)-(B) illustrates comparison of HerDox and Dox intracellulartranslocation and targets in live cells. MDA-MB-435 cells were incubatedwith HerDox or free Dox (0.5 uM) at 37° C. Live (unfixed) cells wereimaged by brightfield and fluorescence, as depicted in FIG. 15 (A), orDIC and confocal fluorescence, as depicted in FIG. 14 (B), microscopy.Dox is indicated by red or magenta pseudocolor.

FIG. 15 illustrates HerDox trafficking in breast cancer cells. Cellsincubated with HerDox at 37° C. were fixed at the indicated time pointsand processed for immunofluorescence using an antibody against HerPBK10.Images were captured using confocal microscopy under fluorescence andbrightfield. Green, HerPBK10; Red, Dox. n, nucleus Bar, ˜8 microns.

FIG. 16 illustrates HerPBK10 binding to MDA-MB-435 cells in human serumfrom HER2+ or HER2− breast cancer patients. Cells were treated withHerPBK10 (1.2 ug/well) in media containing human serum from each of 5HER2+ breast cancer patients or age matched HER2− controls, bothobtained pre-chemotherapy treatment. Cells were processed for ELISAusing an antibody directed at HerPBK10. Control (C) wells receivingHerPBK10 in media containing bovine serum without or with 100× molarexcess competitive ligand inhibitor (+Her) are indicated by open bars.Patient sera were provided by the WCRI tissue bank at Cedars-SinaiMedical Center. N=3 wells per treatment.

FIG. 17 (A)-(B) illustrates relative cell surface HER subunit levels andcytotoxicity on cell types. FIG. 17 (A) depicts a graph of relative cellsurface HER subunit levels as measured by ELISA. Cells were incubatedwith anti-HER subunit antibodies followed by HRP-conjugated secondaryantibodies using standard procedures. Relative cell numbers weremeasured by crystal violet staining and quantified by measuring crystalviolet absorbance at 590 nm. Relative subunit levels are reported as theELISA signal of each cell population normalized by the relative cellnumber, or Abs 450 nm/590 nm. FIG. 17 (B) depicts toxicity to cellsdisplaying differential HER2. Cytotoxicities from a range of HerDoxdoses were assessed on each cell line by metabolic assay and confirmedby crystal violet stain. CD50 values shown in log scale were determinedby non-linear regression analyses of HerDox dose curves using ascientific graphing program and confirmed using a calculator. Therelative HER2 level of each cell line is shown next to each CD50 value.

FIG. 18 illustrates optimization of HerPBK10. The delivery capacity ofthe modified protein, HerPBrgdK10 was tested in the context of anonviral gene transfer complex, and delivery efficiency assessed bytransgene (luciferase) expression in MDA-MB-453 human breast cancercells. *, P<0.005 compared to equivalent concentration of HerPBK10, asdetermined by 2-tailed T test. The figure demonstrates that theinvention is in no way limited to HerPBK10 as various mutations may beintroduced that will improve targeting, receptor binding, cell entryand/or intracellular trafficking of the protein.

FIG. 19 illustrates a graph demonstrating that DS-oligo length does notaffect Dox incorporation into the targeted complex. The graphdemonstrates that there is no appreciable difference in Doxincorporation using either 30 or 48 base pair duplexes.

FIG. 20 (A)-(B) illustrates HerDox is toxic to glioma cells. FIG. 20 (A)depicts HER immunofluorescence on U251 human glioma cells. Images werecaptured using laser scanning fluorescence confocal microscopy. FIG. 20(B) depicts a graph of HerDox vs. Dox toxicity to U251 cells.Significant differences were determined by 2-tailed tests.

DETAILED DESCRIPTION

The invention is based on a novel delivery system including aself-assembling complex for targeting chemical agents to cells. It isbelieved to be advantageous because, among other things, it isself-assembling and less expensive to produce relative to conventionalsystems via large scale fermentation. It is capable of targetingdiseased cells in vitro and in vivo, avoids heart tissue (wheredesirable to do so), and binds and penetrates into target cells.Furthermore, the complex is assembled non-covalently (i.e., without theneed for chemical coupling of, for instance, a chemotherapeutic to atargeted carrier) and it uses a small nucleic acid carrier as a bridgeto assemble the drug with the targeted protein carrier vehicle.

As will be readily appreciated by those of skill in the art, theinvention may have application in a wide variety of fields, includingvarious fields of medicine and the diagnosis, prognosis and treatment ofdisease. In one embodiment, the invention provides a mechanism for thetreatment of cancer by enabling the targeted delivery ofchemotherapeutic agents to cancer cells. In other embodiments, theinventive system may be used to target other types of cells and therebydeliver other chemical agents as may be desired. In another embodiment,the invention provides a mechanism for the imaging of particular cellsor tissues, by the targeted delivery of imaging agents to such tissuesor cells (e.g., cancer cells). Suitable imaging agents will be readilyrecognized by those of skill in the art, and may be used in connectionwith, for example, magnetic resonance imaging of cancer cells withcontrast agents.

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, AdvancedOrganic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley& Sons (New York, N.Y. 2001); and Sambrook and Russel, MolecularCloning: A Laboratory Manual 3rd ed., Cold Spring Harbor LaboratoryPress (Cold Spring Harbor, N.Y. 2001), provide one skilled in the artwith a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

“Beneficial results” may include, but are in no way limited to,preventing, reducing, preventing the increase of and inhibiting theproliferation or growth of cancer cells or tumors. Beneficial resultsmay also refer to curing the cancer and prolonging a patient's life orlife expectancy.

“Cancer” and “cancerous” refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of cancer include, but are not limited to, ovariancancer, breast cancer, colon cancer, lung cancer, prostate cancer,hepatocellular cancer, gastric cancer, pancreatic cancer, cervicalcancer, liver cancer, bladder cancer, cancer of the urinary tract,thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer,and brain cancer.

“Curing” cancer includes degrading a tumor such that a tumor cannot bedetected after treatment. The tumor may be reduced in size or becomeundetectable, for example, by atrophying from lack of blood supply or bybeing attacked or degraded by one or more components administeredaccording to the invention.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be included within the scope of this term.

“Pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

“Prevention” as used herein refers to efforts undertaken to hinder thedevelopment or onset of a condition or cancer condition even if theeffort is ultimately unsuccessful.

“Condition” as used herein refers to an illness or physical ailment.

“Therapeutically effective amount” as used herein refers to that amountwhich is capable of achieving beneficial results in a patient withcancer. A therapeutically effective amount can be determined on anindividual basis and will be based, at least in part, on considerationof the physiological characteristics of the mammal, the type of deliverysystem or therapeutic technique used and the time of administrationrelative to the progression of the disease.

“Treatment” and “treating,” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to achieve beneficial results even if the treatment is ultimatelyunsuccessful.

“Tumor,” as used herein refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

“Chemotherapeutic agent” as used herein refers to agents with thecapability to destroy, kill, hinder the growth of, and/or otherwise havea deleterious effect on cancer cells or tumors. These may include, butare in no way limited to, alkylating agents (e.g., busulfan, cisplatin,carboplatin, chlorambucil, cyclophosphamide, ifosfamide, dacarbazine,mechlorethamine, melphalan, and temozolomide), nitrosoureas (e.g.,streptozocin, carmustine, and lomustine), anthracyclines and relateddrugs (e.g., doxorubicin, epirubicin, idarubicin, and mitoxantrone),topoisomerase I and II inhibitors (e.g., topotecan, irinotecan,etoposide and teniposide), and mitotic inhibitors (e.g., taxanes such aspaclitaxel and docetaxel, and vinca alkaloids such as vinblastine,vincristine, and vinorelbine). Other chemotherapeutic agents will beunderstood by those of skill in the art and can be used in connectionwith alternate embodiments of the present invention by exercise ofroutine effort.

As used herein, “intercalating” refers to the ability to insert into anexisting structure, such as a polynucleotide sequence.

As used herein, “Her” refers to a segment obtained from the receptorbinding domain of heregulin-α, which binds to HER2/HER3 or HER2/HER4subunit heterodimers. As used herein, “PB” refers to a penton basesegment that normally mediates cell binding, entry, and cytosolicpenetration of adenovirus serotype 5 during the early stages ofinfection. An example of a penton base protein is provided herein asSEQ. ID. NO.: 10. This penton base protein normally has an RGD motif(Arg-Gly-Asp). As used herein, “K10” refers to a decalysine motif thathas the capacity to bind nucleic acids by electrophilic interaction,provided herein as SEQ. ID. NO.: 11. An example of a nucleotide sequencecoding for HerPBK10 is provided herein as SEQ. ID. NO.: 4 with itscomplement strand of SEQ. ID. NO.: 5. Similarly, a point mutation of theRGD motif may be used to create an EGD motif (Glu-Gly-Asp), resulting ina HerPBrgdK10 polypeptide molecule (rather than HerPBK10).

As readily apparent to one of skill in the art, any number ofpolynucleotide sequences or small double-stranded nucleic acids may beused in accordance with various embodiments described herein. Forexample, in one embodiment, SEQ. ID. NO.: 6, SEQ. ID. NO.: 7, SEQ. ID.NO.: 8, SEQ. ID. NO.: 9, or a combination thereof, may be used as apolynucleotide sequence or double stranded nucleic acid.

As known to one of skill in the art, any number of targeting ligands maybe used in accordance with various embodiments described herein. Forexample, PB itself may act as a targeting ligand of PBK10 when targetingintegrins such as α_(v)β₃. As known by one of skill in the art,integrins are overly expressed in various types of metastatic tumors.Thus, in conjunction with various embodiments described herein, PBK10may be used to target metastatic tumors and cells with a high expressionof integrins.

The inventive delivery system includes a complex that can beadministered to a mammal by various routes of administration, whereuponit hones in on target cells (e.g., cancer cells) to deliver moleculessuch as imaging agents or therapeutic agents into the cells. In oneembodiment, the complex provides for delivery of therapeutic agents tocancer cells while sparing normal, healthy cells. Current methods oftargeted delivery therapy fail due to the many off-target effects of thetherapy and use of chemical modification strategies that impairtherapeutic activity. One advantage of the inventive delivery system isits targeting effects and retention of delivered agents' therapeuticactivity.

As shown in FIG. 1, an embodiment of the inventive delivery systemincludes three components that self-assemble into one targetedconjugate. The first component (“Unit A”) is a unique cell-penetratingprotein that can target and penetrate a particular type of cell(s). Itincludes a ligand (receptor binding domain), a membrane penetrationdomain, and a DNA binding domain. The second component (“Unit B”) is asmall nucleic acid (e.g., a double-stranded oligonucleotide) that bindsto Unit A via electrostatic interactions. The third component (“Unit C”)is a chemical agent that can bind Unit B via intercalation interactions.In one embodiment of the present invention, the type of cell is a cancercell, and the chemical agent is a chemotherapeutic agent. In anotherembodiment of the present invention, the type of cell is HER2+ breastcancer cells, and the chemical agent is Dox, or a pharmaceuticallyequivalent thereof.

In various embodiments, the inventive delivery system can beincorporated into a pharmaceutical composition, which may be formulatedfor delivery via any route of administration. “Route of administration”may refer to any administration pathway known in the art, including butnot limited to aerosol, nasal, oral, transmucosal, transdermal orparenteral. “Parenteral” refers to a route of administration that isgenerally associated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or in the form of lyophilized powders.

The pharmaceutical compositions according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” as used herein refers to a pharmaceuticallyacceptable material, composition, or vehicle that is involved incarrying or transporting a compound of interest from one tissue, organ,or portion of the body to another tissue, organ, or portion of the body.For example, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulating, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for producing hard gelatin capsule forms. When a liquid carrieris used, the preparation will be in the form of a syrup, elixir,emulsion or an aqueous or non-aqueous suspension. Such a liquidformulation may be administered directly p.o. or filled into a softgelatin capsule.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to, the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

Typical dosages of the inventive delivery system and, more specifically,the therapeutic agents (e.g., chemotherapeutic agents), particularlyDox, and/or the imaging agents delivered by it can be in the rangesrecommended by the manufacturer where known therapeutic compounds orimaging agents are used, and also as indicated to the skilled artisan bythe in vitro responses or responses in animal models. The actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method.

The invention also relates to methods of treating diseases byadministration to a mammal in need thereof of a therapeuticallyeffective amount of the delivery system of the invention including atherapeutic agent appropriate to treat the disease. In one embodiment ofthe invention, the disease is cancer and the therapeutic agent is achemotherapeutic agent. In another embodiment of the invention, thedisease is breast cancer and/or HER2+ breast cancer, and the therapeuticagent is Dox.

The invention also relates to methods of diagnosing and/or prognosing adisease in a mammal by administering to the mammal an effective amountof the delivery system of the invention including an imaging agentappropriate to enable the imaging of cells and/or tissues relevant tothe disease. In one embodiment of the invention, the disease is cancerand an imaging agent is delivered to image cancerous cells or tissue. Inanother embodiment of the invention, the disease is breast cancer and/orHER2+ breast cancer. The methods may include administration of thedelivery system with a suitable imaging agent and the use ofconventional imaging techniques to thereafter image the target tissue orcells and thereby diagnose and/or prognose the disease condition.

In still further embodiments of the present invention, theaforementioned methods may be used in concert to, for example, imagecells or tissues relevant to a disease and then treat the disease. Forinstance, in one embodiment of the present invention, an imaging agentmay be delivered with the inventive delivery system; enabling thediagnosis of HER2+ breast cancer. The inventive delivery system may thenbe utilized to deliver a chemotherapeutic agent (e.g., Dox, orpharmaceutically equivalent thereof) to the HER2+ breast cancer cells.

The present invention is also directed to a kit to treat cancer,including, but in no way limited to, breast cancer and more particularlyHER2+ breast cancer. The kit is useful for practicing the inventivemethod of treating such conditions. The kit is an assemblage ofmaterials or components, including at least one of the components of theinventive delivery system. Thus, in some embodiments, the kit containsthe various components of the inventive delivery system. In otherembodiments, the kit contains all components of the inventive deliverysystem with the exception of the chemotherapeutic agent to be deliveredtherewith.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments areconfigured for the purpose of treating the aforementioned conditions ina subject in need of such treatment. The kit may be configuredparticularly for the purpose of treating mammalian subjects. In anotherembodiment, the kit is configured particularly for the purpose oftreating human subjects. In further embodiments, the kit is configuredfor veterinary applications, for use in treating subjects such as, butnot limited to, farm animals, domestic animals, and laboratory animals.

Other embodiments are configured for the purpose of imaging particularcells or tissues in a subject in whom the imaging of such cells ortissues is desirable. The kit may be configured particularly for thepurpose of imaging cells or tissues in mammalian subjects. In anotherembodiment, the kit is configured particularly for the purpose ofimaging cells or tissues in human subjects, including, but in no waylimited to, breast cancer cells and more particularly HER2+ breastcancer cells. In further embodiments, the kit is configured forveterinary applications, for use in imaging cells and tissues insubjects such as, but not limited to, farm animals, domestic animals,and laboratory animals. In other embodiments, the kit contains allcomponents of the inventive delivery system with the exception of theimaging agent to be delivered therewith.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to treat a disease condition (e.g., cancer) or to imageparticular cells or tissues in a subject. Optionally, the kit alsocontains other useful components such as diluents, buffers,pharmaceutically acceptable carriers, syringes, catheters, applicators,pipetting or measuring tools, bandaging materials or other usefulparaphernalia as will be readily recognized by those of skill in theart.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example, the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging materials employed in the kit are those customarilyutilized in treatment of pituitary disorders and/or tumors and/orcancer. As used herein, the term “package” refers to a suitable solidmatrix or material such as glass, plastic, paper, foil, and the like,capable of holding the individual kit components. Thus, for example, apackage can be one or more glass vials used to contain suitablequantities of the components of the inventive delivery system in anunassembled, a partially assembled, or a completely assembled form. Thepackaging material generally has an external label which indicates thecontents and/or purpose of the kit and/or its components.

EXAMPLES

The following example is provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Targeted Delivery of Chemotherapeutic to HER2+ Breast CancerCells

The inventive technology was tested on HER2+ breast cancer cells invitro and in vivo. As illustrated in FIG. 2, to engineer the inventionto target HER2+ breast cancer, Unit A includes a protein calledHerPBK10, which can be generated by the methods described in L. K.Medina-Kauwe et al., Gene Ther., 8:1753-1761 (2001), incorporated byreference herein in its entirety. HerPBK10 contains the receptor bindingdomain of heregulin fused to the cell penetrating adenovirus penton baseprotein modified by a carboxy (C)-terminal decalysine. The ‘Her’ segmentof HerPBK10 is obtained from the receptor binding domain of heregulin-α,which binds specifically to HER2/HER3 or HER2/HER4 subunit heterodimers.Although heregulin interacts directly with HER3 or HER4, but not HER2,ligand affinity is greatly enhanced by HER2. Thus, tumor cells thatover-express HER2 (i.e., HER2+ tumor cells) are believed to be goodcandidates for heregulin-directed targeting.

The membrane penetrating activity of the adenovirus serotype 5 (Ad5)penton base protein is incorporated into the ‘PB’ segment of HerPBK10 tofacilitate penetration into target cells. The ‘K10’ segment includes tenlysine residues, whose positive charge can facilitate the transport ofnegatively charged molecules, such as nucleic acids, by electrophilicinteraction.

Unit B includes two complementary oligonucleotides annealed together toform a small double-stranded nucleic acid. Unit C is comprised of thechemotherapy agent, Dox.

The three components are assembled together in two steps by incubationat room temperature. In step 1, the Unit B DNA is incubated with theUnit C Dox to form a DNA-Dox assembly (i.e., Unit B+Unit C). This formsby intercalation of the Dox molecules in between the DNA base pairs. Instep 2, the DNA-Dox assembly is incubated with HerPBK10 to form a finalcomplex called HerDox (i.e., Unit A+Unit B+Unit C). This interaction isformed by the electrophilic binding of the negatively charged DNAphosphate backbone to the positively charged polylysine tail ofHerPBK10.

FIG. 3 illustrates a schematic of the operation of the inventivedelivery system in this particular embodiment thereof, and FIG. 11 (B)illustrates its successful application, in vivo, as compared withconventional administration of Dox. Through use of the inventivedelivery system, delivery of Dox was targeted to cancerous cells;relatively higher quantities of the drug were delivered to the cancerouscells as compared with conventionally delivered Dox, and relativelylower quantities of the drug reached non-target healthy tissues.

Example 2 HerDox is Highly Stable During Assembly

HerDox consists of three components: Dox; a small double-strandednucleic acid (which is directly responsible for carrying Dox); and thetargeted protein, HerPBK10. HerDox is assembled in two steps. First, Doxis mixed with the DNA to form a DNA-Dox pair by DNA intercalation. Then,the DNA-Dox pair is mixed with HerPBK10 to form HerDox by electrophilicinteraction. To separate DNA-Dox from free Dox, the mixture underwentultrafiltration centrifugation. The inventors found that >95% of the Doxadded to the DNA did not release from the DNA during the ultrafiltrationspin, indicating high retention of the drug even during a high speedspin (FIG. 4 (A)). The absorbance spectra of retentate and filtrate fromthis spin confirm that the absorbance maximum of retentate coincideswith unfiltered Dox, whereas no such absorbance is detectable in thefiltrate (FIG. 4 (B)). The retentate was then incubated with HerPBK10and the resulting HerDox complex was separated from free components byhigh performance liquid chromatography (HPLC) size exclusion separation.Here the inventors show that the Dox absorbance mostly co-eluted withHerPBK10 (FIG. 5), as confirmed by SDS-PAGE of elution fractions (FIG.5).

Example 3 HerDox is Highly Stable During Storage and in Serum

The inventors tested the stability of HerDox over 12 days underdifferent storage temperatures: 4° C., room temperature, or 37° C. Oneach day, a sample underwent ultrafiltration, then filtrates andretentates were measured to determine whether any Dox was released fromthe complex. At 4° C., 100% of the product remained intact up to 12days, and, interestingly, room temperature and 37° C. appeared toenhance the incorporation of the drug into the HerDox product (FIGS. 6(A) and 6(B)). Altogether, these findings suggest that HerDox remainsstable and does not release Dox after prolonged storage under differenttemperatures. The inventors also examined HerDox stability inserum-containing media at 37° C. HerDox immobilized on nickel sepharose(via the HerPBK10 histidine tag) was incubated at 37° C. in complete(i.e. 10% fetal bovine serum-containing) media (to mimic tissue cultureconditions) for different time periods before the beads were pelletedand supernatants measured for Dox release. Dox retention in theconjugate was also assessed by eluting the conjugate from the beads ateach time point. The inventors observed that the serum produced nosignificant release of Dox from the conjugate, which would be detectedby an increase in Dox filtrate absorbance in the ‘(+) serum’ samples(FIG. 6 (B), upper panel), and that the Dox was completely retained bythe conjugate at each time point (FIG. 6 (B), lower panel).

Example 4 HerDox Produces Targeted Toxicity Whereas Dox Alone does not

The inventors compared the effect of HerDox or Dox alone at equivalentdosages (0.5 uM with respect to Dox concentration) on HER2+ and HER2−breast cancer cells in separate dishes. By three days, HerDox reducedHER2+ cell numbers by over 75% whereas HER2− cell survival wasunaffected (FIG. 7). Equivalent concentrations of Dox alone reduced bothHER2+ and HER2− cells by the same order of magnitude (FIG. 7). Thesefindings emphasize the importance of targeting by showing that theuntargeted drug affects nontarget (i.e. HER2−) cells. Importantly, theprotein carrier, HerPBK10, alone at the equivalent concentration ofprotein in HerDox (0.1 uM) had no effect on either cell line (FIG. 7),including a lack of proliferation induction. To test receptor targeting,the inventors used free heregulin ligand (Her or eHRG) as a competitiveinhibitor. The free ligand completely inhibited cell killing by HerDox(FIG. 8), showing that HerDox bound and entered cells via the heregulinreceptors. As a final in vitro challenge, the inventors tested whetherHerDox induces toxicity specifically to HER2+ cells in a mixed cultureof HER2+ and HER2− breast cancer cells. To do this, the inventorsproduced a HER2− cell line tagged with green fluorescent protein (GFP)for cell identification in the mixed culture (FIG. 9 (A)). The inventorsfound that HerDox nearly completely reduced non-GFP (HER2+) cellproliferation whereas GFP (HER2−) cell growth was not altered (FIG. 9(B)). Altogether, these findings indicate that HerDox has the capacityto preferentially target toxicity to HER2+ cells. Importantly, all ofthese experiments were performed in complete (i.e. serum-containing)media, thus indicating that HerDox can target cells despite the presenceof serum proteins. Moreover, the preferential cell killing of HER2+cells in a mixed culture of HER2+ and HER2− cells implies that afterdeath and lysis of the target cells, the Dox released into those cellsis incapable of continuing to induce toxicity to HER2− cells. To furtherconfirm the stability of HerDox in cell culture, HerDox was recoveredfrom culture media in separate experiments at indicated time points andelectrophoresed on an agarose gel, which was then illuminated by UV todetect Dox and stained with Coomassie blue to detect HerPBK10 protein(FIG. 9 (C)). As the gel does not retain free Dox, loss of Doxfluorescence over time would indicate that the conjugate released Dox.In serum-containing media, no such loss from HerDox is detectable. Inserum-free conditions, it would appear that Dox fluorescence decreasedby 1 h, however Coomassie blue staining shows that the decreasedfluorescence is due to less sample loaded into the gel lane.Co-migration of HerPBK10 bands further verified that the conjugatesremained intact in the cell media. Together with the serum-stabilityassay described earlier, these findings show that the conjugate remainsintact during extended incubation in cell culture (which routinelycontains at least 10% serum).

Example 5 GFP-Her Provides an Index of In Vivo Targeting

To get a sense of the targeting ability of the ligand in vivo andestablish an index of in vivo targeting, the inventors used a greenfluorescent protein (GFP)-tagged ligand (GFP-Her. Importantly, thisligand is identical to the ‘Her’ domain of HerPBK10. We establishedHER2+ tumors in 6-8 week female nude mice via bilateral flank injectionsof MDA-MB-435 cells. When the tumors reached 250-300 mm³ (˜3-4 weeksafter tumor cell implant), 3 nmoles of GFP-Her was injected via the tailvein. Mock injected mice received saline alone. Indicated tissues wereharvested at 3.5 h after injection and imaged for GFP using a XenogenIVIS three-dimensional small-animal in vivo imaging system (Xenogen,Alameda, Calif.). Preferential accumulation of GFP fluorescence wasdetected in the tumors over the other tissues (FIG. 10). Low tonegligible levels of fluorescence were detected in the liver and muscle,while GFP fluorescence was undetectable in the other tissues, includingthe heart (FIG. 10). Tissues from mock-treated animals showed nofluorescence.

Example 6 HerDox Targets HER2+ Breast Cancer Cells In Vivo

Dox emits a red fluorescence upon appropriate wavelength excitation,which can be used to detect biodistribution after systemic delivery ofHerDox. Mice bearing 4-week old tumors (˜700-800 mm³) received a singletail vein injection of Dox or HerDox (0.008 mg/mL with respect to Doxconc) and images of live mice captured in real time, or oforgans/tissues harvested at ˜3 h postinjection were acquired using acustomized macro-illumination and detection system. Fluorescence wasevident throughout the body at 10 min after HerDox injection, thenquickly accumulated at the tumors by 20 min and remained detectable inthe tumors up to 100 min after injection (FIG. 11 (A)). Tissues andtumors harvested at ˜3 h after HerDox injection showed intensefluorescence in the tumors while substantially lower levels offluorescence were detectable in the liver (FIG. 11 (B)). Somefluorescence was barely detectable in the kidneys while other tissues,including the heart, spleen, lungs, and skeletal muscle, did not exhibitany fluorescence. In contrast, tissues harvested from mice injected withthe equivalent dose of Dox exhibited detectable fluorescence in theliver, tumor, and kidneys. Lower levels of fluorescence were alsodetectable in the lungs and skeletal muscle.

To assess in vivo tumor toxicity, mice bearing 3-4 week bilateral flanktumors began receiving daily tail vein injections of Dox or HerDox(0.004 mg/kg with respect to Dox conc), HerPBK10 alone (at equivalentprotein concentration to HerDox) or saline for 7 consecutive days.Tumors were measured throughout tumor growth, beginning 2 weeks beforetail vein injections, and show that while Dox slows tumor growth, HerDoxessentially prevented tumor growth while HerPBK10 alone and saline hadno effect (FIG. 12 (A)). No appreciable weight loss over time wasobserved in either treated or control mice (FIG. 12 (B)).

At 25 days following injections, tumors and organs were harvested andprocessed for histochemistry. It is established that Dox can induceacute and long-term cardiotoxicity, therefore, the inventors examinedthe hearts of mice treated with HerDox or Dox. Hearts from Dox treatedmice appeared slightly enlarged and dilated relative to the hearts fromHerDox and saline-treated mice (not shown), suggestive of the dilatedcardiomyopathy associated with Dox toxicity. Myocardia fromsaline-treated mice exhibited normal cardiac morphology, whereas thethose from Dox-treated mice exhibited focal degeneration, myofibrillarloss, increased cytosoplasmic vacuolization, and nuclear condensation ordissolution, typifying Dox-induced cardiotoxicity, whereas themyocardium from HerDox-treated mice, showed similar morphology to thesaline-treated mice (FIG. 12 (C)). In agreement with these findings,echocardiograms obtained to assess cardiac function in treated mice showsigns of Dox-induced dysfunction that is not detectable inHerDox-treated mice: whereas Dox induces modest to marked reductions instroke volume, cardiac output, and left ventricular internal dimensionand volume, HerDox has no effect on these measurements and appearedsimilar to mock (saline)-treated mice (FIG. 12(D)).

To assess the feasibility of measuring in vivo stability, the inventorsincubated HerDox (at 0.12 mg/mL final Dox conc) or free Dox atequivalent concentration in freshly collected whole blood from mice andincubated the mixtures at 37° C. up to 1 h. As the anticoagulant, 0.5 mMEDTA, was present in the blood collection, parallel samples wereincubated at 37° C. in EDTA alone. Samples representing input HerDox(before incubation in blood) were incubated at 37° C. in HEPES-bufferedsaline. All samples were then centrifuged through 10K MW cutoff filtersand Dox fluorescence measured in retentates and filtrates (SpectraMax M2from Molecular Devices). The results show that there is no detectableloss of Dox from the conjugate, as evidenced by lack of detectableincrease in filtrate fluorescence of HerDox, especially in comparison tothe HerDox incubated in HBS or EDTA, or free Dox (FIG. 13). Likewise,there is no appreciable loss of Dox from HerDox retentates from samplesincubated in blood compared to those incubated in saline or EDTA alone(FIG. 13). Taken together with the bioimaging results, these findingsshow that HerDox remains intact in blood and retains stability in vivowhile in transit toward the tumor target.

Example 7 HerDox Mechanism: Dox Release in Cytoplasm & Accumulation inNucleus

HerPBK10 alone does not induce cell death (FIG. 7), therefore it is thedelivery of Dox into the cell that facilitates cell killing by HerDox.To understand the mechanism of HerDox-mediated tumor cell death, theinventors examined HER2+ cells microscopically after treatment withHerDox, using Dox fluorescence to detect intracellular location. Earlyafter administration (at 0 min of uptake), HerDox appears mostly at thecell periphery, indicating that the conjugate is bound at the cellsurface but not yet internalized (FIG. 14 (A)). In contrast, free Dox isalready found inside the cell at the nuclear periphery (FIG. 14 (A)). At60 min, when the majority of heregulin-targeted proteins have enteredcells, Dox has accumulated in the nucleus, similar to free Dox (FIG. 14(A)). These dynamics, in addition to earlier targeting results, supporta receptor-mediated HerDox entry mechanism. Even the intranuclearpattern of Dox when delivered by HerPBK10 differs from free Dox. Whereasuntargeted Dox accumulates in the cell nucleus, HerDox preferentiallyaccumulates in nucleolar structures with some cytoplasmic fluorescencestill visible (FIG. 14 (B)).

To determine whether Dox remains attached to HerPBK10 during uptake, theinventors used immuno-fluorescence against HerPBK10. At 15 min ofuptake, HerPBK10 mostly colocalizes with Dox, suggesting that asubstantial population of HerDox is still intact, though some nuclearaccumulation of Dox is already visible (FIG. 15). At 30 and 60 min,increasing levels of Dox accumulate in the nucleus while the majority ofHerPBK10 remains in the cytoplasm (FIG. 15). In fixed cells, nucleolaraccumulation was not detectable as in the live cells (FIG. 14 (B)).Altogether, these findings show that HerPBK10 delivers Dox into the celland releases the Dox intracellularly where it undergoes nuclearaccumulation, consistent with the mechanism of delivery (FIG. 11 (B)).

Example 8 Human Serum has No Notable Effect on Cell Binding

To determine whether HerPBK10 can compete with circulating ligand thatmay be present in serum, the inventors tested HerPBK10 binding to HER2+breast cancer cells in human serum obtained from HER2+ patients. TheWomen's Cancer Research Institute at Cedars-Sinai occasionally acquireslimited quantities of patient serum, of which sera from HER2+ patientscomprises an even smaller minority. Notably, the human serum used hereis the actual fraction of serum and associated proteins isolated fromcollected whole blood of HER2+ and age-matched HER2− patients. Earlierexperiments demonstrate that HerDox binds cell targets in completemedium containing 10% bovine serum, and that this binding iscompetitively inhibited by excess free ligand. Here, the inventorsreplaced the bovine serum in the routine culturing media with the humanserum obtained from the acquired patient samples to assess whether thehuman serum, especially from HER2+ patients, inhibits cell binding. Theinventors ensured that cells received considerable exposure to the humansera (2 hours, which provides ample time for receptor binding of anycirculating ligand) prior to treatment. Head-to-head comparisons of cellbinding in serum from either HER2+ patients, HER2− patients, or bovineserum show no significant differences (FIG. 16), indicating that thehuman sera tested here did not interfere with HerPBK10 binding to targetcells. Competitive inhibition with 100× heregulin ligand (+Her) confirmsthat the control binding activity is specific to heregulin receptors.

Example 9 HER Subunit Levels and Cytoxicity on Proposed Cell Types

The inventors measured cell surface levels of HER subunits on variouscell lines and types described herein, as previously described levelsmay not reflect the actual levels in the available cells used. Theinventors acquired the indicated cell lines from ATCC and the NIH/NCIand profiled these with respect to HER subunit levels (FIG. 17 (A)). Toassess whether HerDox induces toxicity in accordance to HER2 levels, theinventors selected lines displaying HER2 at relatively high (SKBR3),moderate (MDA-MB-435, MDA-MB-453, HeLa), and low to undetectable(MDA-MB-231) levels, and performed cytotoxicity dose curves. Theinventors observed that HerDox CD50 inversely correlates with cellsurface HER2 level on these selected lines: the cell line displayingrelatively high HER2 shows a relatively higher sensitivity to HerDoxwhereas the cell line displaying low HER2 exhibits low sensitivity, andthe lines displaying intermediate HER2 levels likewise exhibitintermediate sensitivities (FIG. 17 (B); CD50 is shown on a log scale).

TABLE 1 Cytotoxicity on cell lines Example 10 CELL LINE HER2* EC50** (uMHerDox) MDA-MB-231  0.06 ± 0.006 7.2e5 ± 0.11  MDA-MB-435 0.52 ± 0.080.74 ± 0.07 T47D 1.03 ± 0.26 0.64 ± 0.04 SKOV3 1.79 ± 0.19 0.18 ± 0.03*Relative cell surface level (mean ± 1SD) as determined ELISA. N = 3wells. **Concentration (mean ± 1SD) yielding 50% reduction in cellsurvival, as determined by nonlinear regression analyses of HerDox dosecurves. N = 3 treated wells per dose.

Example 11 Optimization of HerPBK10

As the HerPBK10 protein originates from the adenovirus penton baseprotein, whose natural binding targets are alpha-v integrins, theinventors assessed whether mutation of the Arg-Gly-Asp (RGD) integrinbinding motif improves the capacity of the protein to deliver cargo intocells. While previous studies indicate that appendage of the heregulinreceptor binding ligand to the penton base redirects it nearlyexclusively to heregulin receptors (as demonstrated by competitiveinhibition assay), it is possible that HerPBK10 may still co-optintegrin receptors that may redirect the protein to a differentintracellular route or compete for binding sites on the protein itself.Rendering the RGD motif to EGD by point mutation disables integrinbinding. Therefore, the mutant protein, HerPBrgdK10 was produced bearingthis mutation, and tested for gene delivery in comparison to parentalHerPBK10. At equivalent protein concentrations, HerPBrgdK10 exhibitedmoderate (˜1.8-fold) to dramatic (˜18-fold) enhancement of gene transfer(FIG. 18), which may be reflective of enhanced receptor binding orpost-binding activities.

Example 12 DNA Constructs

The inventors used a common 5′ oligonucleotide primer containing thesequence 5′-ATCGAAGGATCCATGCGGCGCGCGGCGATGTAT-3′ (SEQ. ID. NO.: 12) toamplify both wild-type and lysine-tagged penton sequences from a pJM17adenoviral genome template. The sequences of the 3′ primers are PB:5′-GCATCAGAATTCTCAAAAAGTGCGGCTCGATAG-3′ (SEQ. ID. NO.: 1) and PBK105′-CATGAATTCA(TTT)10AAAAGTGCGGCTCGATAGGA-3′ (SEQ. ID. NO.: 2). A BamHIrestriction site was introduced in the 5′ primer and an EcoRIrestriction site was introduced in the 3′ primers for in-frame insertionof both the wild-type and lysine-tagged pentons into the pRSET-Abacterial expression plasmid (Invitrogen, Carlsbad, Calif., USA). Thisplasmid expresses the recombinant protein as an N-terminallyhistidine-tagged fusion for affinity purification by nickel chelateaffinity chromatography.

Polymerase chain reaction (PCR) amplification was used to add a sequenceencoding a short polyglycine linker to the amino (N)-terminus of PBK10.The sequence encoding the linker contains a SacII restriction site foradditional cloning. The heregulin targeting ligand was produced by PCRamplification of the epidermal growth factor (EGF)-like domain of theheregulin gene29 using a 5′ oligonucleotide primer containing a BamHIsite and a 3′ primer containing a SacII site for cloning in-frame withPBK10. The targeting ligand was added to the lysinetagged construct tocreate HerPBK10 by ligating the PCR product just N-terminal to PBK10.Construction of Her and GFP-Her have been previously described(Medina-Kauwe L K, et al., BioTechniques 2000, 29: 602-609). HerK10 wascreated by PCR amplification of the Her construct using a 5′ Her primer(Medina-Kauwe L K, et al., BioTechniques 2000, 29: 602-609) and a 3′oligonucleotide primer containing the sequence5′-ATGAATTCA(TTT)10AGATCTACTTCCACCACTTCCACC-3′ (SEQ. ID. NO.: 3).

Example 13 DS-Oligo Length does not Affect Dox Incorporation into theTargeted Complex

Ds-oligo duplexes were formed from complimentary 30 bp sequences, LLAA-5(SEQ. ID. NO.: 6) and LLAA-3 (SEQ. ID. NO.: 7) or 48 bp sequences,BglIIHis-5 (SEQ. ID. NO.: 8) and BglIIHis-3 (SEQ. ID. NO.: 9). Dox wasadded to each set of annealed duplexes at either 1:10, 1:20, or 1:40molar ratio duplex:Dox (at a final Dox concentration of either 20, 40,or 80 uM) in 10 mM Tris/HCl buffer, pH 8.0, for 30 minutes at roomtemperature. The mixtures were then centrifuged through ultrafiltrationmembranes (Microcon Ultracel YM10; Millipore) at 10,000×g to separatefree Dox from incorporated Dox. Retentates and filtrates were collectedseparately, and absorbances of each measured at 480 nm using aSpectraMax M2 plate reader (Molecular Devices). The results (FIG. 19)show that there is no appreciable difference in Dox incorporation usingeither 30 or 48 bp duplexes.

Example 14 HerDox is Toxic to Glioma Cells

U251 human glioma cells were assessed for HER subunit levels bynon-permeabilizing immunohistochemistry and found to display relativelymarked levels of cell surface HER2, HER3, and HER4 (FIG. 20 (A)).

U251 cells growing in dishes were incubated with either HerDox or Dox(at either 0.5 uM or 1 uM) in the culture medium for 4 hours at 37° C.,5% C02, after which fresh complete medium was added to increase thefinal culture volume approximately four-fold, and the cells maintainedat 37° C., 5% C02 for four days. Cells were trypsinized and counted onthe last day.

The inventors' results show that HerDox exhibits 8-10 times moretoxicity to U251 cells than the equivalent concentration of Dox, andlikewise 10× less HerDox elicits the same toxicity as Dox (FIG. 20 (B)).

Example 15 Summary

These studies indicate that a stable non-covalent conjugate can assembleand direct a well-established chemotherapy drug to target cells inserum. Delivery is mediated via the heregulin receptor, as free ligandcompetitively inhibits delivery. Importantly, the inventors havedemonstrated that toxicity can be targeted to HER2+ cells in a mixedcell culture and in vivo. These studies show that the carrier protein,HerPBK10, is capable of mediating targeted toxicity and that drugconjugates non-covalently linked to this carrier can be assembled anddelivered with apparently little to no premature release or nonspecifictoxicity.

While the description above refers to particular embodiments of thepresent invention, it should be readily apparent to people of ordinaryskill in the art that a number of modifications may be made withoutdeparting from the spirit thereof. The presently disclosed embodimentsare, therefore, to be considered in all respects as illustrative and notrestrictive. One skilled in the art will recognize many methods andmaterials similar or equivalent to those described herein, which couldbe used in the practice of the present invention. Indeed, the presentinvention is in no way limited to the methods and materials described.For example, various agents may be delivered in conjunction withembodiments described herein and the invention should not be merelylimited to Dox or chemotherapy agents. Similarly, various motifs couldbe used interchangeably with or in addition to those described hereinand the invention should not be construed as limited to only polylysinemotifs and/or RGD motifs. Finally, as recognized by one of skill in theart, the invention can be applied to any number of conditions, disordersand/or diseases where it is advantageous to target delivery of an agentto a cell and/or cell nucleus and the present invention should not beconstrued in any way as limited to the treatment of breast cancer.

The invention claimed is:
 1. A method of treating a disease in anindividual, comprising: administering a therapeutically effective amountof a drug delivery molecule to the individual, the drug deliverymolecule comprising: a polypeptide sequence adapted to target andpenetrate a type of cell, a nucleic acid sequence bound to thepolypeptide sequence via electrostatic interactions, and a chemicalagent non-covalently linked to the nucleic acid sequence.
 2. The methodof claim 1, wherein the disease is breast cancer.
 3. The method of claim1, wherein the chemical agent is a chemotherapeutic agent.
 4. The methodof claim 1, wherein the chemical agent is doxorubicin.
 5. The method ofclaim 1, wherein the polypeptide sequence comprises a targeting ligand,an endosomolytic domain, or a polylysine motif.
 6. The method of claim1, wherein the individual is a human.
 7. The method of claim 1, whereinthe type of cell is a glioma cell.
 8. The method of claim 1, wherein thepolypeptide sequence comprises PBK10.
 9. The method of claim 1, whereinthe disease is metastatic cancer.
 10. The method of claim 1, wherein thedisease is cancer.
 11. The method of claim 1, wherein the type of cellis a breast cancer cell.
 12. The method of claim 1, wherein the type ofcell is a HER2+ breast cancer cell.
 13. The method of claim 1, whereinthe polypeptide sequence comprises a targeting ligand.
 14. The method ofclaim 13, wherein the targeting ligand comprises Her.
 15. The method ofclaim 1, wherein the polypeptide sequence comprises a penton base or avariant thereof.
 16. The method of claim 15, wherein the penton basecomprises an EGD motif in place of an RGD motif.
 17. The method of claim15, wherein the polypeptide sequence further comprises a targetingligand.
 18. The method of claim 17, wherein the targeting ligandcomprises Her.
 19. The method of claim 1, wherein the polypeptidesequence comprises a polylysine motif.
 20. The method of claim 19,wherein the polypeptide sequence further comprises a penton base or avariant thereof.
 21. The method of claim 20, wherein the polypeptidesequence further comprises a targeting ligand.
 22. The method of claim21, wherein the targeting ligand comprises Her.
 23. The method of claim1, wherein the nucleic acid sequence comprises a sequence according toSEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:
 9. 24. Themethod of claim 1, wherein the nucleic acid sequence is a doublestranded nucleic acid sequence comprising a first nucleic acid moleculehaving a sequence according to SEQ ID NO: 6, and a second nucleic acidmolecule having a sequence according to SEQ ID NO:
 7. 25. The method ofclaim 1, wherein the chemical agent intercalates with the nucleic acidsequence.
 26. The method of claim 1, wherein the polypeptide sequencecomprises a targeting ligand, a penton base or a variant thereof, and apolylysine motif.
 27. The method of claim 1, wherein the polypeptidesequence comprises HerPBK10.
 28. The method of claim 27, wherein thenucleic acid sequence is a double-stranded nucleic acid sequence. 29.The method of claim 28, wherein the chemical agent comprisesdoxorubicin.
 30. The method of claim 29, wherein the doxorubicinintercalates with the double stranded nucleic acid sequence.
 31. Themethod of claim 28, wherein the double-stranded nucleic acid sequencecomprises a first nucleic acid molecule having a sequence according toSEQ ID NO: 6, and a second nucleic acid molecule having a sequenceaccording to SEQ ID NO:
 7. 32. The method of claim 31, wherein thechemical agent comprises doxorubicin.
 33. The method of claim 32,wherein the doxorubicin intercalates with the double stranded nucleicacid sequence.